U.S. patent number 9,856,509 [Application Number 14/267,276] was granted by the patent office on 2018-01-02 for fluorescent substrates for determining lysine modifying enzyme activity.
This patent grant is currently assigned to The Broad Institute, Inc.. The grantee listed for this patent is The Broad Institute, Inc.. Invention is credited to Edward Holson, Florence F. Wagner, Yan-Ling Zhang.
United States Patent |
9,856,509 |
Zhang , et al. |
January 2, 2018 |
Fluorescent substrates for determining lysine modifying enzyme
activity
Abstract
The invention relates to a compound of Formula I:
F.sub.1--X.sub.1-L.sub.1-X.sub.2--P.sub.1--X.sub.3-G.sub.1 (Formula
I).
Inventors: |
Zhang; Yan-Ling (Lexington,
MA), Holson; Edward (Newton, MA), Wagner; Florence F.
(Ashland, MA) |
Applicant: |
Name |
City |
State |
Country |
Type |
The Broad Institute, Inc. |
Cambridge |
MA |
US |
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Assignee: |
The Broad Institute, Inc.
(Cambridge, MA)
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Family
ID: |
48192840 |
Appl.
No.: |
14/267,276 |
Filed: |
May 1, 2014 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20140335550 A1 |
Nov 13, 2014 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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PCT/US2012/063377 |
Nov 2, 2012 |
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61628562 |
Nov 2, 2011 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07D
311/82 (20130101); C12Q 1/34 (20130101); C07K
5/10 (20130101); C12Q 1/37 (20130101); C09K
11/06 (20130101); C07K 7/06 (20130101); C09K
2211/1007 (20130101); C09K 2211/1088 (20130101); G01N
2333/98 (20130101) |
Current International
Class: |
C07K
7/06 (20060101); C12Q 1/37 (20060101); C09K
11/06 (20060101); C07K 5/10 (20060101); C12Q
1/34 (20060101); C07D 311/82 (20060101); C12N
9/14 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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2459976 |
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Nov 2009 |
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GB |
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02/12267 |
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Feb 2002 |
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WO |
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2010/092381 |
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Aug 2010 |
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WO |
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2010/121023 |
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Oct 2010 |
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WO |
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2011/019393 |
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Feb 2011 |
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WO |
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Other References
Wegener, D; Wirsching, F; Riester, D; and Schwienhorst, A "A
Fluorogenic Histone Deacetylase Assay Well Suited for
High-Throughput Activity Screening" Chemistry & Biology, vol.
10, 61-68, Jan. 2003. cited by examiner .
Hu, M., et al., "Supporting Information: Multi-Color, One-and
Two-Photon Imaging of Enzymatic Activities in Live Cells with
Fluorescently Quenched Activity-Based Probes (qABPs)," J. Am. Chem.
Soc., 133(31): 12009-12020 (2011). cited by applicant .
Jiskoot, W., et al., "Preparation and Application of a
Fluorescein-Labeled Peptide for Determining the Affinity Constant
of a Monoclonal Antibody-Hapten Complex by Fluorescence
Polarization," Anal. Biochem., 196(2): 421-426 (1991). cited by
applicant .
Dumelin, C. E., et al., "A Portable Albumin Binder from a
DNA-Encoded Chemical Library," Angew. Chem. Int. Ed., 47(17):
3196-3201 (2008). cited by applicant .
Ikeda, M., et al., "Development of a DNA-binding TEMPO derivative
for evaluation of nuclear oxidative stress and its application in
living cells," Free Radical Biology and Medicine, 49(11): 1792-1797
(2010). cited by applicant .
Wollack, J. W., et al., "Multifunctional Prenylated Peptides for
live Cell Analysis," J. Am. Chem. Soc., 131(21): 7293-7303 (2009).
cited by applicant .
De Waart, D. R., et al., "Hepatic Transport Mechanisms of
Cholyl-L-Lysyl-Fluorescein," J. Pharmacol. Exp. Therapeutics,
334(1): 78-86 (2010). cited by applicant .
Mills, C. O., et al., "Different pathways of canalicular secretion
of sulfated and non-sulfated fluorescent bile acids: a study in
isolated hepatocyte couplets and TR-rats," J. Hepatology, 31(4):
678-684 (1999). cited by applicant .
Lohse, J., et al., "Fluorescein-Conjugated Lysine Monomers for
Solid Phase Synthesis of Fluorescent Peptides and PNA Oligomers,"
Bioconj. Chem., 8(4): 503-509 (1997). cited by applicant.
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Primary Examiner: Tate; Chris R
Assistant Examiner: Kosar; Aaron J
Attorney, Agent or Firm: Elmore Patent Law Group, P.C. Hoda;
Mahreen Elmore; Carolyn
Parent Case Text
RELATED APPLICATIONS
This application is a continuation of International Application No.
PCT/US2012/063377, which designated the United States and was filed
on Nov. 2, 2012, published in English, which claims the benefit of
U.S. Provisional Application No. 61/628,562, filed on Nov. 2, 2011.
The entire teachings of the above applications are incorporated
herein by reference.
Claims
What is claimed is:
1. A compound of Formula I or a salt thereof:
F.sub.1--X.sub.1-L.sub.1-X.sub.2--P.sub.1--X.sub.3-G.sub.1 (Formula
I); wherein: F.sub.1 is a fluorophore; L.sub.1 is alkylene,
substituted alkylene, alkenylene, substituted alkenylene,
alkynylene, or substituted alkynylene; P.sub.1 is a peptide
containing a lysine residue, wherein the peptide can act as a
substrate of a lysine deacetylase; G.sub.1 is coumarin, methyl
coumarin, or N-methyl-3-phenylpropanamide; each of X.sub.1,
X.sub.2, and X.sub.3 is independently a direct bond, --O--, --S--,
--C(O)--, --C(O)--NR.sub.100--, --C(S)--, --C(S)--NR.sub.100--,
--C(O)O--, --NR.sub.100-- and --S(O).sub.2--; and each R.sub.100 is
independently hydrogen, alkyl, substituted alkyl, aryl or
substituted aryl.
2. The compound according to claim 1, wherein F.sub.1 is a
fluorescein-based fluorophore.
3. The compound according to claim 2, wherein F.sub.1 is 6-carboxy
fluorescein (6-FAM), 5-carboxy fluorescein (5-FAM), or fluorescein
isothiocyanate (FITC).
4. The compound according to claim 1, wherein L.sub.1 is a
C.sub.1-C.sub.10 alkylene group.
5. The compound according to claim 1, wherein P.sub.1 is LGK(Ac),
TGGK(Ac)APR (SEQ ID NO: 4), LGKGGAK(Ac) (SEQ ID NO: 5), TSPQPKK(Ac)
(SEQ ID NO: 6), SPQPKK(Ac) (SEQ ID NO: 7), PQPKK(Ac) (SEQ ID NO:
8), TSRHK(Ac) (SEQ ID NO: 9), RGK(Ac), LGK(COCF.sub.3),
TGGK(COCF.sub.3)APR (SEQ ID NO: 10), LGKGGAK(COCF.sub.3) (SEQ ID
NO: 11), TSPQPKK(COCF.sub.3) (SEQ ID NO: 12), SPQPKK(COCF.sub.3)
(SEQ ID NO: 13), PQPKK(COCF.sub.3) (SEQ ID NO: 14),
TSRHK(COCF.sub.3) (SEQ ID NO: 15), RGK(COCF.sub.3), RHKK(Ac) (SEQ
ID NO: 16), QPKK(Ac) (SEQ ID NO: 17), RHKK(COCF.sub.3) (SEQ ID NO:
18), QPKK(COCF.sub.3) (SEQ ID NO: 19), RHKK (SEQ ID NO: 20), QPKK
(SEQ ID NO: 21), LGK, TGGKAPR (SEQ ID NO: 22), LGKGGAK (SEQ ID NO:
23), TSPQPKK (SEQ ID NO: 24), SPQPKK (SEQ ID NO: 25), PQPKK (SEQ ID
NO: 26), TSRHK (SEQ ID NO: 27) or RGK.
6. The compound according to claim 1, wherein X.sub.1 is selected
from --O--, --C(O)NH--, --C(O)-- and --C(O)O--.
7. The compound according to claim 1, wherein X.sub.2 is selected
from --O--, --C(O)NH--, --C(O)-- and --C(O)O--.
8. The compound according to claim 1, wherein X.sub.3 is selected
from --O--, --C(O)NH--, --C(O)-- and --C(O)O--.
9. The compound according to claim 1, wherein F.sub.1 is selected
from Table A: TABLE-US-00007 TABLE A ##STR00129## ##STR00130##
##STR00131## ##STR00132## ##STR00133## ##STR00134##
##STR00135##
10. The compound of claim 9, wherein F.sub.1 is ##STR00136##
11. The compound of claim 10, wherein P.sub.1 is selected from the
group consisting of: ##STR00137##
12. The compound of claim 11, wherein G.sub.1 is coumarin or methyl
coumarin.
13. The compound of claim 12, wherein G.sub.1 is ##STR00138##
14. The compound according to claim 1, wherein L.sub.1 is selected
from Table B: TABLE-US-00008 TABLE B ##STR00139## ##STR00140##
##STR00141## ##STR00142## ##STR00143## ##STR00144## ##STR00145##
##STR00146##
15. The compound according to claim 1, wherein P.sub.1 is selected
from Table C: TABLE-US-00009 TABLE C ##STR00147## ##STR00148##
##STR00149## ##STR00150## ##STR00151## ##STR00152## ##STR00153##
##STR00154## ##STR00155## ##STR00156## ##STR00157## ##STR00158##
##STR00159## ##STR00160## ##STR00161## ##STR00162## ##STR00163##
##STR00164## ##STR00165## ##STR00166## ##STR00167## ##STR00168##
##STR00169## ##STR00170## ##STR00171## ##STR00172## ##STR00173##
##STR00174## ##STR00175## ##STR00176## ##STR00177## ##STR00178##
##STR00179## ##STR00180## ##STR00181## ##STR00182## ##STR00183##
##STR00184## ##STR00185## ##STR00186## ##STR00187## ##STR00188##
##STR00189## ##STR00190## ##STR00191## ##STR00192## ##STR00193##
##STR00194## ##STR00195## ##STR00196## ##STR00197## ##STR00198##
##STR00199## ##STR00200## ##STR00201## ##STR00202##
##STR00203##
wherein each R.sub.105 and R.sub.106 is independently hydrogen,
--C(O)--C.sub.1-C.sub.6 alkyl, --C(S)--C.sub.1-C.sub.6 alkyl,
--C(O)-substituted C.sub.1-C.sub.6 alkyl, --C(S)-substituted
C.sub.1-C.sub.6 alkyl, C(O)-aryl, --C(S)-aryl, --C(O)-substituted
aryl, --C(S)-substituted aryl, --S(O)--C.sub.1-C.sub.6 alkyl,
--S(O).sub.2--C.sub.1-C.sub.6 alkyl, alkyl, substituted
C.sub.1-C.sub.6 alkyl, aryl or substituted aryl.
16. The compound of claim 15, wherein P.sub.1 is selected from the
group consisting of: ##STR00204##
17. The compound according to claim 1, wherein G.sub.1 is
##STR00205##
18. The compound of claim 1, wherein the lysine residue of P.sub.1
is acetylated or trifluoroacetylated.
19. A compound selected from Table E or a salt thereof:
TABLE-US-00010 TABLE E ##STR00206## Broad Substrate D ##STR00207##
Broad Substrate C ##STR00208## Broad Substrate B ##STR00209## Broad
Substrate A
20. A method for determining lysine deacetylase activity comprising
the step of incubating a lysine deacetylase with a compound
according to claim 1 and monitoring the modification of a lysine
residue of said compound over time.
21. The method according to claim 20, wherein said step of
monitoring the modification of a lysine residue of said compound
comprises the step of monitoring the fluorescence of said compound
or the product resulting from the modification the lysine
residue.
22. The method according to claim 21, wherein the modification of
the lysine residue is deacetylation of an acetylated lysine residue
or detrifluoroacetylation of a trifluoroacetylated lysine
residue.
23. The method according to claim 20, wherein said step of
monitoring the modification of the lysine residue over time
comprises separating the product of resulting from the modification
of the lysine residue from the compound using a microfluidic
mobility shift technology.
24. A method for determining histone deacetylase (HDAC) activity
comprising the step of incubating an HDAC with a compound according
to claim 1 and determining the activity of the HDAC enzyme.
25. The method according to claim 24, wherein said HDAC is an
isoform of HDAC selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or
SIRT 1, 2, 3, 4, 5, 6 or 7.
26. The method according to claim 24, wherein said step of
determining the activity of the HDAC enzyme comprises the
monitoring of the release of G.sub.1.
27. A method for determining histone acetyltransferase activity
comprising the step of incubating a histone acetyl transferase with
a compound according to claim 1 and monitoring the modification of
a lysine residue of said compound over time.
28. The method according to claim 27, wherein said step of
determining the activity of the histone acetyltransferase enzyme
comprises the step of monitoring the fluorescence of the compound
or the product of the modification of the lysine residue.
29. The method according to claim 27, wherein said step of
determining the activity of the histone acetyltransferase enzyme
comprises the monitoring of the release of G.sub.1.
Description
BACKGROUND
Lysine acetylation and deacetylation play important roles in the
modulation of chromatin topology and the regulation of gene
transcription. Lysine deacetylases and histone deacetylases in
particular are proven drug targets for cancer and also potential
targets for neurological diseases. A commonly used assay for HDAC
activity is a trypsin coupled fluorogenic assay. This indirect
endpoint assay is simple and applicable for high throughput
screening. However, it is limited in its ability to continuously
monitor enzyme activity due to protein's stability in the presence
of trypsin. In addition, trypsin inhibitors may impair the assay
results. Recently, Caliper microfluidic lab-on-a-chip technology
has been used to measure HDAC activity and characterize HDAC
inhibitors. This assay directly follows the separated
fluorophore-labeled substrate and product using FAM labeled
acetylated peptide. Advantages of this direct assay include its
ability to continuously monitor enzyme activity and the ability to
determine enzyme activity in the absence of trypsin or other
proteases which may degrade protein components within the assay.
Interference from fluorescent compounds is minimized in screening.
This assay is currently limited for a few HDACs such as HDAC3 and 6
due to the lack of efficient HDAC substrates for other HDAC
isoforms including HDAC1 and 2. As such, substrates used to
determine the activity of all HDAC isoforms using the microfuidic
lab-on-chip technology is needed.
SUMMARY OF THE INVENTION
The invention relates to fluorescent conjugates of Formula I:
F.sub.1--X.sub.1-L.sub.1-X.sub.2--P.sub.1--X.sub.3-G.sub.1 (Formula
I)
wherein F.sub.1 is a fluorophore; preferably a fluoresceine based
fluorophore; more preferably, 6-carboxy fluorescein (6-FAM);
L.sub.1 is alkyl, substituted alkyl, alkenyl, substituted alkenyl,
alkynyl, substituted alkynyl, --O--, --S--,
--[C(R.sub.10)(R.sub.11)].sub.t--, --N(R.sub.10)--,
--N(R.sub.10)[C(R.sub.10)(R.sub.11)].sub.t,
--O[C(R.sub.10)(R.sub.11)].sub.t--,
--O[C(R.sub.10)(R.sub.11)C(R.sub.10)(R.sub.11)O].sub.u-- or
--S[C(R.sub.10)(R.sub.11)].sub.t-aliphatic or substituted
aliphatic; wherein t is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24; u is an integer
between 1 and 500; each R.sub.10 and R.sub.11 is independently
hydrogen, halogen, --OR.sub.20, --SR.sub.20, --NR.sub.20R.sub.21,
--CF.sub.3, --CN, --NO.sub.2, --N.sub.3, --C(O)OR.sub.20,
--C(O)R.sub.20, --C(O)NR.sub.20R.sub.21, acyl, alkoxy, substituted
alkoxy, alkylamino, substituted alkylamino, dialkylamino,
substituted dialkylamino, substituted or unsubstituted alkylthio,
substituted or unsubstituted alkylsulfonyl, aliphatic, substituted
aliphatic, aryl or substituted aryl; alternatively two of R.sub.10
and R.sub.11 groups together with the atoms to which they are
attached and any intervening atoms may form an additional
optionally substituted, 3, 4, 5, 6 or 7 membered ring; wherein each
R.sub.20 and R.sub.21 is independently hydrogen, aliphatic,
substituted aliphatic, aromatic or substituted aromatic; P.sub.1 is
a peptide or protein that can act as a substrate for an enzyme that
modifies a lysine or substituted lysine residue, preferably a
lysine deacetylase, which include but are not limited to
NAD+-dependent sirtuins (SIRT) and zinc dependent histone
deacetylase (HDAC), or lysine acetyltransferase, preferably a
histone acetyltransferase; P.sub.1 is preferably a one, two, three,
four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen,
fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty,
twenty-one or twenty-two amino acid peptide containing a lysine
residue wherein the side chain of the lysine residue is
unsubstituted, acetylated or trifluoroacetylated, preferably
comprising of natural amino acids, with optional additional
substitution; more preferably, a two, three four five or six amino
acid peptide comprising natural amino acids with optional
additional substitution; more preferably said two, three, four,
five or six amino acid peptide is a peptide comprised of natural
amino acids, containing an unsubstituted side-chain lysine residue
(K) that can be acetylated or a side-chain acetylated lysine
residue (K(Ac)) or a side-chain trifluoroacetylated lysine residue
(K(COCF.sub.3) that can be deacetylated by a HDAC enzyme, or
trifluoroacetyl substituted lysine residue that can act as a
substrate for HDAC enzyme; more preferably, P.sub.1 is a peptide
having the sequence of AA.sub.1-AA.sub.2-AA.sub.3-AA.sub.4-Lys(Ac)
(SEQ ID NO: 2), AA.sub.1-AA.sub.2-AA.sub.3-AA.sub.4-Lys(COCF.sub.3)
(SEQ ID NO: 3), or AA.sub.1-AA.sub.2-AA.sub.3-AA.sub.4-Lys (SEQ ID
NO: 1) wherein each AA.sub.1, AA.sub.2 and AA.sub.3 is absent or a
natural or unnatural amino acid, and AA.sub.4 is a natural or
unnatural amino acid; G.sub.1 is hydrophobic group; Preferably,
G.sub.1 is an optionally substituted alkyl, optionally substituted
alkenyl, optionally substituted alkynyl, optionally substituted
aryl, optionally substituted heteroaryl group, an alkyl group
substituted with an optionally substituted aryl or heteroaryl
group, an alkenyl group substituted with an optionally substituted
aryl or heteroaryl group or a natural or unnatural amino acid;
Wherein each X.sub.1, X.sub.2, and X.sub.3 is independently a
direct bond, --O--, --S--, --C(O)--, --C(O)--NR.sub.100--,
--C(S)--, --C(S)--NR.sub.100--, --C(O)O--, --NR.sub.100-- and
--S(O).sub.2--; wherein R.sub.100 is hydrogen, alkyl, substituted
alkyl, aryl or substituted aryl.
The invention further relates to the use of compound of Formula I
for the determination of histone deacetylase activity. The
compounds of Formula I can be used to determine the activity of
HDAC isoforms 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or SIRT 1, 2, 3, 4,
5, 6 or 7. In one embodiment, these substrates allow the
measurement of activity of full length or truncated variants of
histone deacetylases (HDAC 1-11 or SIRT 1-7) and their
corresponding complexes with microfluidic lab-on-chip technology.
In addition, these substrates can be used for screening HDAC
inhibitors, studying mechanism of inhibition and profiling their
selectivity. Without being bound to any theory it is postulated
that peptide, P.sub.1, with C-terminal G.sub.1 is a much more
efficient HDAC substrate than the conjugate without G.sub.1. By
conjugating G.sub.1 to P.sub.1 its specific activity can be
increased, for example by more than 100 fold, for acetylated lysine
containing peptide substrate. The increased activity can lead to
the reduced use of enzymes to reach the desired amount of substrate
conversion.
BRIEF DESCRIPTION OF THE DRAWINGS
The foregoing and other objects, features and advantages of the
invention will be apparent from the following more particular
description of preferred embodiments of the invention, as
illustrated in the accompanying drawings in which like reference
characters refer to the same parts throughout the different views.
The drawings are not necessarily to scale, emphasis instead being
placed upon illustrating the principles of the invention.
FIG. 1: Comparison of HDAC 1,2,3 activity with Broad Substrate A
vs. commercial substrate (H218).
FIG. 2: Comparison of Coumarine effect on substrate
specificity/efficiency.
FIG. 3: % Substrate conversion by HDAC 1-9 using Broad Substrate A
and Broad Substrate B.
FIG. 4: Determination of IC.sub.50 (.mu.M) values for control
compounds with no preincubation on HDAC 1-9 using Broad
Substrates.
FIG. 5: Comparison of known HDAC inhibitors IC.sub.50 (.mu.M)
values with no preincubation (SAHA, CI-994, BRD6929) using Broad
Substrate A vs. commercial substrate.
FIG. 6A is a graph which shows a time course of substrate
conversion (%) with HDAC 1 in the presence of various
concentrations of SAHA (Example 6).
FIG. 6B is a graph which shows substrate conversion (%) over time
in a reversibility assay for SAHA (Example 6). The red circles
represent compound dilution.
FIG. 7A is a graph which shows a time course of substrate
conversion (%) with HDAC 1 in the presence of various
concentrations of CI-994 (Example 6).
FIG. 7B is a graph which shows substrate conversion (%) over time
in a reversibility assay for CI-994(Example 6). The red circles
represent compound dilution.
FIG. 8: Schematic diagram showing the use of microfluidic mobility
shift assay to determine substrate modification via charged based
separation of substrate from product, where an HDAC enzyme
deacetylates an acetylated-lysine-containing substrate.
DETAILED DESCRIPTION OF THE INVENTION
The invention relates to fluorescent conjugates of Formula I:
F.sub.1--X.sub.1-L.sub.1-X.sub.2--P.sub.1--X.sub.3-G.sub.1 (Formula
I) Wherein F.sub.1 is a fluorophore; preferably a fluoresceine
based fluorophore; more preferably, FAM; L.sub.1 is alkyl,
substituted alkyl, alkenyl, substituted alkenyl, alkynyl,
substituted alkynyl, --O--, --S--,
--[C(R.sub.10)(R.sub.11)].sub.t--, --N(R.sub.10)--,
--N(R.sub.10)[C(R.sub.10)(R.sub.11)].sub.t--,
--O[C(R.sub.10)(R.sub.11)].sub.t--,
--O[C(R.sub.10)(R.sub.11)C(R.sub.10)(R.sub.11)O].sub.u-- or
--S[C(R.sub.10)(R.sub.11)].sub.t-aliphatic or substituted
aliphatic; Wherein t is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24; u is an integer
between 1 and 500; Each R.sub.10 and R.sub.11 is independently
hydrogen, halogen, --OR.sub.20, --SR.sub.20, --NR.sub.20R.sub.21,
--CF.sub.3, --CN, --NO.sub.2, --N.sub.3, --C(O)OR.sub.20,
--C(O)R.sub.20, --C(O)NR.sub.20R.sub.21, acyl, alkoxy, substituted
alkoxy, alkylamino, substituted alkylamino, dialkylamino,
substituted dialkylamino, substituted or unsubstituted alkylthio,
substituted or unsubstituted alkylsulfonyl, aliphatic, substituted
aliphatic, aryl or substituted aryl; alternatively two of R.sub.10
and R.sub.11 groups together with the atoms to which they are
attached and any intervening atoms may form an additional
optionally substituted, 3, 4, 5, 6 or 7 membered ring; Wherein each
R.sub.20 and R.sub.21 is independently hydrogen, aliphatic,
substituted aliphatic, aromatic or substituted aromatic; P.sub.1 is
a peptide or protein that can act as a substrate to a lysine
deacetylase, preferably, a histone deacetylase (HDAC); P.sub.1 is
preferably a one, two, three, four, five, six, seven, eight, nine,
ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen,
seventeen, eighteen or nineteen amino acid peptide containing a
lysine residue wherein the side chain of the lysine residue is
unsubstituted, acetylated or trifluoroacetylated, more preferably
comprising of natural amino acids, with optional additional
substitution; preferably, a two, three or four amino acid peptide
comprising natural amino acids with optional additional
substitution; preferably a peptide containing a side-chain
acetylated lysine residue (K(Ac)) that can be deacetylated by a
HDAC enzyme, or trifluoroacetyl substituted lysine residue
(K(COCF.sub.3)) that can act as a substrate for HDAC enzyme, or an
unsubstituted side-chain lysine residue (K) that can be acetylated;
more preferably, P.sub.1 is a peptide having the sequence of
AA.sub.1-AA.sub.2-AA.sub.3-AA.sub.4-Lys(Ac)--,
AA.sub.1-AA.sub.2-AA.sub.3-AA.sub.4-Lys or
AA.sub.1-AA.sub.2-AA.sub.3-AA.sub.4-Lys(COCF.sub.3)-- wherein each
AA.sub.1, AA.sub.2 and AA.sub.3 is absent or a natural or unnatural
amino acid, and AA.sub.4 is a natural or unnatural amino acid;
preferably each AA.sub.1, AA.sub.2, AA.sub.3 or AA.sub.4 is
independently selected from Isoleucine (I), Alanine (A), Leucine
(L), Asparagine (N), Lysine (K), Aspartic acid (D), Methionine (M),
Cysteine (C), Phenylalanine (P), Glutamic acid (E), Threonine (T),
Glutamine (Q), Tryptophan (W), Glycine (G), Valine (V), Proline
(P), Serine (S), Tyrosine (Y), Arginine (R) and Histidine (H),
G.sub.1 is hydrophobic group that can be cleaved from X.sub.3 or
P.sub.1 by a protease, preferably trypsin; Preferably, this an
optionally substituted alkyl, optionally substituted alkenyl,
optionally substituted alkynyl, optionally substituted aryl,
optionally substituted heteroaryl group, an alkyl group substituted
with an optionally substituted aryl or heteroaryl group, an alkenyl
group substituted with an optionally substituted aryl or heteroaryl
group or a natural or unnatural amino acid; Wherein each X.sub.1,
X.sub.2, and X.sub.3 is independently a direct bond, --O--, --S--,
--C(O)--, --C(O)--NR.sub.100--, --C(S)--, --C(S)--NR.sub.100--,
--C(O)O--, --NR.sub.100-- and --S(O).sub.2--; wherein R.sub.100 is
hydrogen, alkyl, substituted alkyl, aryl or substituted aryl. In a
preferred embodiment, F.sub.1 is FAM. In a preferred embodiment,
L.sub.1 is an alkyl or C.sub.1-C.sub.10 alkyl group. In a preferred
embodiment, P.sub.1 is a peptide selected from LGK(Ac) or
TGGK(Ac)APR (SEQ ID NO: 4), LGKGGAK(Ac) (SEQ ID NO: 5), TSPQPKK(Ac)
(SEQ ID NO: 6), SPQPKK(Ac) (SEQ ID NO: 7), PQPKK(Ac) (SEQ ID NO:
8), TSRHK(Ac) (SEQ ID NO: 9), RGK(Ac), LGK(COCF.sub.3) or
TGGK(COCF.sub.3)APR (SEQ ID NO: 10), LGKGGAK(COCF.sub.3) (SEQ ID
NO: 11), TSPQPKK(COCF.sub.3) (SEQ ID NO: 12), SPQPKK(COCF.sub.3)
(SEQ ID NO: 13), PQPKK(COCF.sub.3) (SEQ ID NO: 14),
TSRHK(COCF.sub.3) (SEQ ID NO: 15), RGK(COCF.sub.3), RHKK(Ac) (SEQ
ID NO: 16), QPKK(Ac) (SEQ ID NO: 17), RHKK(COCF.sub.3) (SEQ ID NO:
18), QPKK(COCF.sub.3) (SEQ ID NO: 19), RHKK (SEQ ID NO: 20), QPKK
(SEQ ID NO: 21), LGK, TGGKAPR (SEQ ID NO: 22), LGKGGAK (SEQ ID NO:
23), TSPQPKK (SEQ ID NO: 24), SPQPKK (SEQ ID NO: 25), PQPKK (SEQ ID
NO: 26), TSRHK (SEQ ID NO: 27) or RGK; more preferably LGK, LGK(Ac)
or LGK(COCF.sub.3). In a preferred embodiment, G.sub.1 is methyl
coumarin or coumarin or N-methyl-3-phenylpropanamide. In a
preferred embodiment, X.sub.1 is selected from --O--, --C(O)NH--,
--C(O)-- and --C(O)O--. In a preferred embodiment, X.sub.2 is
selected from --O--, --C(O)NH--, --C(O)-- and --C(O)O--. In a
preferred embodiment, X.sub.3 is selected from --O--, --C(O)NH--,
--C(O)-- and --C(O)O--. In a preferred embodiment, F.sub.1 is
selected from Table A:
TABLE-US-00001 TABLE A ##STR00001## ##STR00002## ##STR00003##
##STR00004## ##STR00005## ##STR00006## ##STR00007##
Wherein a is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In a preferred
embodiment, L.sub.1 is selected from Table B:
TABLE-US-00002 TABLE B ##STR00008## ##STR00009## ##STR00010##
##STR00011## ##STR00012## ##STR00013## ##STR00014## ##STR00015##
##STR00016## ##STR00017## ##STR00018## ##STR00019## ##STR00020##
##STR00021## ##STR00022## ##STR00023## ##STR00024##
##STR00025##
Wherein each t is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24; i is 1,
2, 3, 4, 5 or 6; j is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; u is an
integer between 1 and 500 or between 1 and 10 or between 1 and 20
or between 1 and 100 or between 1 and 300; Each R.sub.101 and
R.sub.102 is independently selected from hydrogen, halogen,
--OR.sub.20, --SR.sub.20, --NR.sub.20R.sub.21, --CF.sub.3, --CN,
--NO.sub.2, --N.sub.3, --C(O)OR.sub.20, --C(O)R.sub.20,
--C(O)NR.sub.20R.sub.21, acyl, alkoxy, substituted alkoxy,
alkylamino, substituted alkylamino, dialkylamino, substituted
dialkylamino, substituted or unsubstituted alkylthio, substituted
or unsubstituted alkylsulfonyl, aliphatic, substituted aliphatic,
aryl or substituted aryl --S(O).sub.2R.sub.100,
--S(O).sub.3R.sub.100, --S(O).sub.3H, Alternatively, two R.sub.101
and R.sub.102 groups together with the atom or atoms to which they
are attached may form one, two or three rings with optional
additional substitution. In a preferred embodiment, P.sub.1 is
selected from Table C:
TABLE-US-00003 TABLE C ##STR00026## ##STR00027## ##STR00028##
##STR00029## ##STR00030## ##STR00031## ##STR00032## ##STR00033##
##STR00034## ##STR00035## ##STR00036## ##STR00037## ##STR00038##
##STR00039## ##STR00040## ##STR00041## ##STR00042## ##STR00043##
##STR00044## ##STR00045## ##STR00046## ##STR00047## ##STR00048##
##STR00049## ##STR00050## ##STR00051## ##STR00052## ##STR00053##
##STR00054## ##STR00055## ##STR00056## ##STR00057## ##STR00058##
##STR00059## ##STR00060## ##STR00061## ##STR00062## ##STR00063##
##STR00064## ##STR00065## ##STR00066## ##STR00067## ##STR00068##
##STR00069## ##STR00070## ##STR00071## ##STR00072## ##STR00073##
##STR00074## ##STR00075## ##STR00076## ##STR00077## ##STR00078##
##STR00079## ##STR00080## ##STR00081## ##STR00082##
wherein each R.sub.105 and R.sub.106 is independently hydrogen,
--C(O)alkyl, --C(S)alkyl, --C(O)substituted alkyl,
--C(S)substituted alkyl, C(O)aryl, --C(S)aryl, --C(O)substituted
aryl, --C(S)substituted aryl, --S(O)alkyl, --S(O).sub.2alkyl,
alkyl, substituted alkyl, aryl or substituted aryl. In a preferred
embodiment, G.sub.1 is selected from Table D:
TABLE-US-00004 TABLE D ##STR00083## ##STR00084## ##STR00085##
##STR00086## ##STR00087## ##STR00088## ##STR00089## ##STR00090##
##STR00091## ##STR00092## ##STR00093## ##STR00094## ##STR00095##
##STR00096## ##STR00097## ##STR00098## ##STR00099## ##STR00100##
##STR00101## ##STR00102## ##STR00103## ##STR00104## ##STR00105##
##STR00106## ##STR00107## ##STR00108##
Each v and w is independently 0, 1, 2, 3 or 4; In a preferred
embodiment, the invention relates to the compounds of Table E.
TABLE-US-00005 TABLE E ##STR00109## Broad Substrate D ##STR00110##
Broad Substrate C ##STR00111## Broad Substrate B ##STR00112## Broad
Substrate A ##STR00113## Broad Substrate E
The invention further comprises a method for determining Lysine
modifying enzyme activity by incubating the enzyme with a compound
of Formula I. In one embodiment, the enzyme activity determination
comprise the step of incubating the enzyme with a compound of
Formula I (aka the substrate) and monitoring the product formation
over time.
In one embodiment, the invention relates to a method for
determining an HDAC enzyme activity by incubating an HDAC enzyme
with a compound of Formula I wherein the compound of Formula I has
a substituted lysine residue, preferably acetyl or trifluoroacetyl
substituted lysine residue. In a preferred embodiment, the HDAC
enzyme is an isoform selected from HDAC 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11 or SIRT 1, 2, 3, 4, 5, 6 or 7. In one embodiment, the HDAC
activity determination comprises the step of incubating an HDAC
enzyme with a compound of Formula I (aka the substrate) and
monitoring the product formation over time by fluorescence wherein
the compound of Formula I has a substituted lysine residue,
preferably acetyl or trifluoroacetyl substituted lysine residue. In
one embodiment, the product is separated from the substrate using a
microfluidic mobility shift technology. In one embodiment, the
compound of Formula I is incubated with an HDAC enzyme followed by
incubation with trypsin wherein trypsin can lead to the cleavage of
G.sub.1. In one embodiment, the release of G1 is monitored by
fluorescence and correlated with HDAC activity.
In one embodiment, the invention relates to a method for evaluating
histone acetyltransferase activity comprising the step of
incubating histone acetyltransferase enzyme with a compound of
Formula I, wherein said compound of Formula I contains an
unsubstituted lysine residue, and determining the activity of the
histone acetyltransferase enzyme.
The invention further comprises a method for determining an enzyme
activity comprising the step of incubating a compound that can be
modified by two or more enzymes wherein the first enzyme is lysine
modifying and wherein said compound comprises at least one
fluorescent moiety and a chromophore moiety wherein one of
chromophore moiety can be cleaved by a second enzyme. In a
preferred embodiment the substrate is a compound of claim 1 and the
chromophore is G.sub.1, preferably coumarin. In a preferred
embodiment, the fluorescent moiety is Fluorescein based
fluorophore, preferably FAM, most preferably 6-FAM. In a preferred
embodiment, the chromophore is cleaved following the treatment with
trypsin.
Definitions
Listed below are definitions of various terms used to describe this
invention. These definitions apply to the terms as they are used
throughout this specification and claims, unless otherwise limited
in specific instances, either individually or as part of a larger
group.
The term "aliphatic group" or "aliphatic" refers to a non-aromatic
moiety that may be saturated (e.g., single bond) or contain one or
more units of unsaturation, e.g., double and/or triple bonds. An
aliphatic group may be straight chained, branched or cyclic,
contain carbon, hydrogen or, optionally, one or more heteroatoms
and may be substituted or unsubstituted. In addition to aliphatic
hydrocarbon groups, aliphatic groups include, for example,
polyalkoxyalkyls, such as polyalkylene glycols, polyamines, and
polyimines, for example. Such aliphatic groups may be further
substituted. It is understood that aliphatic groups may include
alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,
substituted alkynyl, and substituted or unsubstituted cycloalkyl
groups as described herein.
The term "acyl" refers to a carbonyl substituted with hydrogen,
alkyl, partially saturated or fully saturated cycloalkyl, partially
saturated or fully saturated heterocycle, aryl, or heteroaryl. For
example, acyl includes groups such as (C.sub.1-C.sub.6) alkanoyl
(e.g., formyl, acetyl, propionyl, butyryl, valeryl, caproyl,
t-butylacetyl, etc.), (C.sub.3-C.sub.6)cycloalkylcarbonyl (e.g.,
cyclopropylcarbonyl, cyclobutylcarbonyl, cyclopentylcarbonyl,
cyclohexylcarbonyl, etc.), heterocyclic carbonyl (e.g.,
pyrrolidinylcarbonyl, pyrrolid-2-one-5-carbonyl,
piperidinylcarbonyl, piperazinylcarbonyl,
tetrahydrofuranylcarbonyl, etc.), aroyl (e.g., benzoyl) and
heteroaroyl (e.g., thiophenyl-2-carbonyl, thiophenyl-3-carbonyl,
furanyl-2-carbonyl, furanyl-3-carbonyl, 1H-pyrroyl-2-carbonyl,
1H-pyrroyl-3-carbonyl, benzo[b]thiophenyl-2-carbonyl, etc.). In
addition, the alkyl, cycloalkyl, heterocycle, aryl and heteroaryl
portion of the acyl group may be any one of the groups described in
the respective definitions. When indicated as being "optionally
substituted", the acyl group may be unsubstituted or optionally
substituted with one or more substituents (typically, one to three
substituents) independently selected from the group of substituents
listed below in the definition for "substituted" or the alkyl,
cycloalkyl, heterocycle, aryl and heteroaryl portion of the acyl
group may be substituted as described above in the preferred and
more preferred list of substituents, respectively.
The term "alkyl" is intended to include both branched and straight
chain, substituted or unsubstituted saturated aliphatic hydrocarbon
radicals/groups having the specified number of carbons. Preferred
alkyl groups comprise about 1 to about 24 carbon atoms
("C.sub.1-C.sub.24"). Other preferred alkyl groups comprise at
about 1 to about 8 carbon atoms ("C.sub.1-C.sub.8") such as about 1
to about 6 carbon atoms ("C.sub.1-C.sub.6"), or such as about 1 to
about 3 carbon atoms ("C.sub.1-C.sub.3"). Examples of
C.sub.1-C.sub.6 alkyl radicals include, but are not limited to,
methyl, ethyl, propyl, isopropyl, n-butyl, tert-butyl, n-pentyl,
neopentyl and n-hexyl radicals.
The term "alkenyl" refers to linear or branched radicals having at
least one carbon-carbon double bond. Such radicals preferably
contain from about two to about twenty-four carbon atoms
("C.sub.2-C.sub.24"). Other preferred alkenyl radicals are "lower
alkenyl" radicals having two to about ten carbon atoms
("C.sub.2-C.sub.10") such as ethenyl, allyl, propenyl, butenyl and
4-methylbutenyl. Preferred lower alkenyl radicals include 2 to
about 6 carbon atoms ("C.sub.2-C.sub.6"). The terms "alkenyl", and
"lower alkenyl", embrace radicals having "cis" and "trans"
orientations, or alternatively, "E" and "Z" orientations.
The term "alkynyl" refers to linear or branched radicals having at
least one carbon-carbon triple bond. Such radicals preferably
contain from about two to about twenty-four carbon atoms
("C.sub.2-C.sub.24"). Other preferred alkynyl radicals are "lower
alkynyl" radicals having two to about ten carbon atoms such as
propargyl, 1-propynyl, 2-propynyl, 1-butyne, 2-butynyl and
1-pentynyl. Preferred lower alkynyl radicals include 2 to about 6
carbon atoms ("C.sub.2-C.sub.6").
The term "cycloalkyl" refers to saturated carbocyclic radicals
having three to about twelve carbon atoms ("C.sub.3-C.sub.12"). The
term "cycloalkyl" embraces saturated carbocyclic radicals having
three to about twelve carbon atoms. Examples of such radicals
include cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.
The term "cycloalkenyl" refers to partially unsaturated carbocyclic
radicals having three to twelve carbon atoms. Cycloalkenyl radicals
that are partially unsaturated carbocyclic radicals that contain
two double bonds (that may or may not be conjugated) can be called
"cycloalkyldienyl". More preferred cycloalkenyl radicals are "lower
cycloalkenyl" radicals having four to about eight carbon atoms.
Examples of such radicals include cyclobutenyl, cyclopentenyl and
cyclohexenyl.
The term "alkylene," as used herein, refers to a divalent group
derived from a straight chain or branched saturated hydrocarbon
chain having the specified number of carbons atoms. Examples of
alkylene groups include, but are not limited to, ethylene,
propylene, butylene, 3-methyl-pentylene, and 5-ethyl-hexylene.
The term "alkenylene," as used herein, denotes a divalent group
derived from a straight chain or branched hydrocarbon moiety
containing the specified number of carbon atoms having at least one
carbon-carbon double bond. Alkenylene groups include, but are not
limited to, for example, ethenylene, 2-propenylene, 2-butenylene,
1-methyl-2-buten-1-ylene, and the like.
The term "alkynylene," as used herein, denotes a divalent group
derived from a straight chain or branched hydrocarbon moiety
containing the specified number of carbon atoms having at least one
carbon-carbon triple bond. Representative alkynylene groups
include, but are not limited to, for example, propynylene,
1-butynylene, 2-methyl-3-hexynylene, and the like.
The term "alkoxy" refers to linear or branched oxy-containing
radicals each having alkyl portions of one to about twenty-four
carbon atoms or, preferably, one to about twelve carbon atoms. More
preferred alkoxy radicals are "lower alkoxy" radicals having one to
about ten carbon atoms and more preferably having one to about
eight carbon atoms. Examples of such radicals include methoxy,
ethoxy, propoxy, butoxy and tert-butoxy.
The term "alkoxyalkyl" refers to alkyl radicals having one or more
alkoxy radicals attached to the alkyl radical, that is, to form
monoalkoxyalkyl and dialkoxyalkyl radicals.
The term "aryl", alone or in combination, means an aromatic system
containing one, two or three rings wherein such rings may be
attached together in a pendent manner or may be fused. The term
"aryl" embraces aromatic radicals such as phenyl, naphthyl,
tetrahydronaphthyl, indane furanyl, quinazolinyl, pyridyl and
biphenyl.
The terms "heterocyclyl", "heterocycle" "heterocyclic" or
"heterocyclo" refer to saturated, partially unsaturated and
unsaturated heteroatom-containing ring-shaped radicals, which can
also be called "heterocyclyl", "heterocycloalkenyl" and
"heteroaryl" correspondingly, where the heteroatoms may be selected
from nitrogen, sulfur and oxygen. Examples of saturated
heterocyclyl radicals include saturated 3 to 6-membered
heteromonocyclic group containing 1 to 4 nitrogen atoms (e.g.,
pyrrolidinyl, imidazolidinyl, piperidino, piperazinyl, etc.);
saturated 3 to 6-membered heteromonocyclic group containing 1 to 2
oxygen atoms and 1 to 3 nitrogen atoms (e.g., morpholinyl, etc.);
saturated 3 to 6-membered heteromonocyclic group containing 1 to 2
sulfur atoms and 1 to 3 nitrogen atoms (e.g., thiazolidinyl, etc.).
Examples of partially unsaturated heterocyclyl radicals include
dihydrothiophene, dihydropyran, dihydrofuran and dihydrothiazole.
Heterocyclyl radicals may include a pentavalent nitrogen, such as
in tetrazolium and pyridinium radicals. The term "heterocycle" also
embraces radicals where heterocyclyl radicals are fused with aryl
or cycloalkyl radicals. Examples of such fused bicyclic radicals
include benzofuran, benzothiophene, and the like.
The term "heteroaryl" refers to unsaturated aromatic heterocyclyl
radicals. Examples of heteroaryl radicals include unsaturated 3 to
6 membered heteromonocyclic group containing 1 to 4 nitrogen atoms,
for example, pyrrolyl, pyrrolinyl, imidazolyl, pyrazolyl, pyridyl,
pyrimidyl, pyrazinyl, pyridazinyl, triazolyl (e.g.,
4H-1,2,4-triazolyl, 1H-1,2,3-triazolyl, 2H-1,2,3-triazolyl, etc.),
tetrazolyl (e.g., 1H-tetrazolyl, 2H-tetrazolyl, etc.), etc.;
unsaturated condensed heterocyclyl group containing 1 to 5 nitrogen
atoms, for example, indolyl, isoindolyl, indolizinyl,
benzimidazolyl, quinolyl, isoquinolyl, indazolyl, benzotriazolyl,
tetrazolopyridazinyl (e.g., tetrazolo[1,5-b]pyridazinyl, etc.),
etc.; unsaturated 3 to 6-membered heteromonocyclic group containing
an oxygen atom, for example, pyranyl, furyl, etc.; unsaturated 3 to
6-membered heteromonocyclic group containing a sulfur atom, for
example, thienyl, etc.; unsaturated 3- to 6-membered
heteromonocyclic group containing 1 to 2 oxygen atoms and 1 to 3
nitrogen atoms, for example, oxazolyl, isoxazolyl, oxadiazolyl
(e.g., 1,2,4-oxadiazolyl, 1,3,4-oxadiazolyl, 1,2,5-oxadiazolyl,
etc.) etc.; unsaturated condensed heterocyclyl group containing 1
to 2 oxygen atoms and 1 to 3 nitrogen atoms (e.g., benzoxazolyl,
benzoxadiazolyl, etc.); unsaturated 3 to 6-membered
heteromonocyclic group containing 1 to 2 sulfur atoms and 1 to 3
nitrogen atoms, for example, thiazolyl, thiadiazolyl (e.g.,
1,2,4-thiadiazolyl, 1,3,4-thiadiazolyl, 1,2,5-thiadiazolyl, etc.)
etc.; unsaturated condensed heterocyclyl group containing 1 to 2
sulfur atoms and 1 to 3 nitrogen atoms (e.g., benzothiazolyl,
benzothiadiazolyl, etc.) and the like.
The term "heterocycloalkyl" refers to heterocyclo-substituted alkyl
radicals. More preferred heterocycloalkyl radicals are "lower
heterocycloalkyl" radicals having one to six carbon atoms in the
heterocyclo radical.
The term "alkylthio" refers to radicals containing a linear or
branched alkyl radical, of one to about ten carbon atoms attached
to a divalent sulfur atom. Preferred alkylthio radicals have alkyl
radicals of one to about twenty-four carbon atoms or, preferably,
one to about twelve carbon atoms. More preferred alkylthio radicals
have alkyl radicals which are "lower alkylthio" radicals having one
to about ten carbon atoms. Most preferred are alkylthio radicals
having lower alkyl radicals of one to about eight carbon atoms.
Examples of such lower alkylthio radicals include methylthio,
ethylthio, propylthio, butylthio and hexylthio.
The terms "aralkyl" or "arylalkyl" refer to aryl-substituted alkyl
radicals such as benzyl, diphenylmethyl, triphenylmethyl,
phenylethyl, and diphenylethyl.
The term "aryloxy" refers to aryl radicals attached through an
oxygen atom to other radicals.
The terms "aralkoxy" or "arylalkoxy" refer to aralkyl radicals
attached through an oxygen atom to other radicals.
The term "aminoalkyl" refers to alkyl radicals substituted with
amino radicals. Preferred aminoalkyl radicals have alkyl radicals
having about one to about twenty-four carbon atoms or, preferably,
one to about twelve carbon atoms. More preferred aminoalkyl
radicals are "lower aminoalkyl" that have alkyl radicals having one
to about ten carbon atoms. Most preferred are aminoalkyl radicals
having lower alkyl radicals having one to eight carbon atoms.
Examples of such radicals include aminomethyl, aminoethyl, and the
like.
The term "alkylamino" denotes amino groups which are substituted
with one or two alkyl radicals. Preferred alkylamino radicals have
alkyl radicals having about one to about twenty carbon atoms or,
preferably, one to about twelve carbon atoms. More preferred
alkylamino radicals are "lower alkylamino" that have alkyl radicals
having one to about ten carbon atoms. Most preferred are alkylamino
radicals having lower alkyl radicals having one to about eight
carbon atoms. Suitable lower alkylamino may be monosubstituted
N-alkylamino or disubstituted N,N-alkylamino, such as
N-methylamino, N-ethylamino, N,N-dimethylamino, N,N-diethylamino or
the like.
The term "substituted" refers to the replacement of one or more
hydrogen radicals in a given structure with the radical of a
specified substituent including, but not limited to: halo, alkyl,
alkenyl, alkynyl, aryl, heterocyclyl, thiol, alkylthio, arylthio,
alkylthioalkyl, arylthioalkyl, alkylsulfonyl, alkylsulfonylalkyl,
arylsulfonylalkyl, alkoxy, aryloxy, aralkoxy, aminocarbonyl,
alkylaminocarbonyl, arylaminocarbonyl, alkoxycarbonyl,
aryloxycarbonyl, haloalkyl, amino, trifluoromethyl, cyano, nitro,
alkylamino, arylamino, alkylaminoalkyl, arylaminoalkyl,
aminoalkylamino, hydroxy, alkoxyalkyl, carboxyalkyl,
alkoxycarbonylalkyl, aminocarbonylalkyl, acyl, aralkoxycarbonyl,
carboxylic acid, sulfonic acid, sulfonyl, phosphonic acid, aryl,
heteroaryl, heterocyclic, and aliphatic. It is understood that the
substituent may be further substituted.
For simplicity, chemical moieties that are defined and referred to
throughout can be univalent chemical moieties (e.g., alkyl, aryl,
etc.) or multivalent moieties under the appropriate structural
circumstances clear to those skilled in the art. For example, an
"alkyl" moiety can be referred to a monovalent radical (e.g.
CH.sub.3--CH.sub.2--), or in other instances, a bivalent linking
moiety can be "alkyl," in which case those skilled in the art will
understand the alkyl to be a divalent radical (e.g.,
--CH.sub.2--CH.sub.2--), which is equivalent to the term
"alkylene." Similarly, in circumstances in which divalent moieties
are required and are stated as being "alkoxy", "alkylamino",
"aryloxy", "alkylthio", "aryl", "heteroaryl", "heterocyclic",
"alkyl" "alkenyl", "alkynyl", "aliphatic", or "cycloalkyl", those
skilled in the art will understand that the terms "alkoxy",
"alkylamino", "aryloxy", "alkylthio", "aryl", "heteroaryl",
"heterocyclic", "alkyl", "alkenyl", "alkynyl", "aliphatic", or
"cycloalkyl" refer to the corresponding divalent moiety.
The terms "halogen" or "halo" as used herein, refers to an atom
selected from fluorine, chlorine, bromine and iodine.
The term natural amino acid includes the following naturally
occurring amino acids: Isoleucine, Alanine, Leucine, Asparagine,
Lysine, Aspartic acid, Methionine, Cysteine, Phenylalanine,
Glutamic acid, Threonine, Glutamine, Tryptophan, Glycine, Valine.
Proline, Selenocysteine, Serine, Tyrosine, Arginine, Histidine,
Ornithine and Taurine. As used herein the modified lysine residues
K(Ac) and K(COCF.sub.3), as part of a peptide or conjugate has the
following structures:
##STR00114##
The terms "compound" "drug", and "prodrug" as used herein all
include pharmaceutically acceptable salts, co-crystals, solvates,
hydrates, polymorphs, enantiomers, diastereoisomers, racemates and
the like of the compounds, drugs and prodrugs having the formulas
as set forth herein.
Substituents indicated as attached through variable points of
attachments can be attached to any available position on the ring
structure.
EXAMPLES
Example 1: Synthesis of
5-(((4S,7S,13S)-4-benzyl-13-isobutyl-3,6,9,12,15-pentaoxo-7-(4-(2,2,2-tri-
fluoroacetamido)butyl)-2,5,8,11,14-pentaazaicosan-20-yl)carbamoyl)-2-(6-hy-
droxy-3-oxo-3H-xanthen-9-yl)benzoic Acid, Broad Substrate D
##STR00115##
The compound depicted above was synthesized according to the
following procedure:
##STR00116## To a solution of
(S)-2-((tert-butoxycarbonyl)amino)-4-methylpentanoic acid (1) (10
g, 43.2 mmol, 1.0 equiv.) in THF (90 mL) was added methyl
2-aminoacetate hydrochloride (2) (3.2 g, 43.2 mmol, 1.0 equiv.),
Et.sub.3N (12.5 g, 124 mmol, 2.9 equiv.) and HATU (16.4 g, 43.2
mmol, 1.0 equiv.). The mixture was stirred at room temperature for
16 h. The reaction was filtered through Celite. The reaction
filtrate was diluted with 100 mL of water and stirred for 15 min.
The suspension was filtered off, rinsed with water and dried to
afford (S)-methyl
2-(2-((tert-butoxycarbonyl)amino)-4-methylpentanamido)acetate (3)
(12 g, 92% yield).
##STR00117## To a solution of (S)-methyl
2-(2-((tert-butoxycarbonyl)amino)-4-methylpentanamido) acetate (3)
(10 g, 33.1 mmol) in 1,4-dioxane (50 mL) was added a 5M solution of
HCl in 1,4-dioxane (50 mL) at room temperature. The reaction was
stirred at room temperature for 16 h. The reaction mixture was
filtered to afford (S)-methyl
2-(2-amino-4-methylpentanamido)acetate hydrochloride (4) as the
filtered solid (7.9 g, 100%).
##STR00118## To a solution of (S)-methyl
2-(2-amino-4-methylpentanamido)acetate hydrochloride (4) (5 g, 21
mmol, 1.0 equiv.) in THF (80 mL) was added
6-((tert-butoxycarbonyl)amino) hexanoic acid (4.8 g, 21 mmol, 1.0
equiv.), HATU (12 g, 31.5 mmol, 1.5 equiv.) and DIPEA (10.74 g,
83.2 mmol, 4.0 equiv.). The reaction was stirred at room
temperature for 18 h. The mixture was then filtered through Celite.
The filtrate was concentrated under reduced pressure and the crude
residue was purified by column chromatography (silica gel,
CH.sub.2Cl.sub.2/MeOH=50/1) to give (S)-methyl
13-isobutyl-2,2-dimethyl-4,11,14-trioxo-3-oxa-5,12,15-triazaheptadecan-17-
-oate (6) as a white solid (5 g, 57% yield).
##STR00119## To a solution of (5)-methyl
13-isobutyl-2,2-dimethyl-4,11,14-trioxo-3-oxa-5,12,15-triazaheptadecan-17-
-oate (6) (5 g, 12 mmol) in THF (50 mL) was added a solution of
LiOH.H.sub.2O (1.25 g, 30 mmol, 2.5 equiv.) in water (50 mL) at
room temperature. After 3 h, the reaction mixture was concentrated,
diluted with water and acidified with a 1N aqueous solution of HCl
to about pH4-5. The mixture was stirred for 15 min and the white
precipitate formed was filtered off, rinsed with water, and dried
to afford
(S)-13-isobutyl-2,2-dimethyl-4,11,14-trioxo-3-oxa-5,12,15-triazaheptadeca-
n-17-oic acid (7) (2 g, 42% yield).
##STR00120## To a solution of
(S)-2-((tert-butoxycarbonyl)amino)-6-(2,2,2-trifluoroacetamido)hexanoic
acid (8) (0.90 g, 2.63 mmol, 1.0 equiv.) in DMF (25 mL) at room
temperature was added (S)-2-amino-N-methyl-3-phenylpropanamide
hydrochloride (0.56 g, 2.63 mmol, 1.0 equiv.), HATU (3.99 g, 10.51
mmol, 4.0 equiv.) and triethylamine (2.13 g, 21.02 mmol, 8.0
equiv.). The reaction was stirred at room temperature for 2 h. A
saturated solution of sodium bicarbonate was added. The product was
extracted with ethyl acetate. The combined organic layers were
washed with water, dried over sodium sulfate, filtered and
concentrated under reduced pressure. The crude residue was purified
by prep-HPLC to afford tert-butyl
((S)-1-(((S)-1-(methylamino)-1-oxo-3-phenylpropan-2-yl)amino)-1-oxo-6-(2,-
2,2-trifluoroacetamido)hexan-2-yl)carbamate (9) (0.86 g, 65% yield)
as a solid.
##STR00121## To a solution of tert-butyl
((S)-1-(((S)-1-(methylamino)-1-oxo-3-phenylpropan-2-yl)amino)-1-oxo-6-(2,-
2,2-trifluoroacetamido)hexan-2-yl)carbamate (9) (0.38 g, 0.87 mmol)
in 1,4-dioxane (10 mL) was added a 5M solution of HCl in
1,4-dioxane (20 mL). The reaction was stirred at room temperature
for 3 h. The mixture was concentrated under reduced pressure to
afford
(S)-2-amino-N--((S)-1-(methylamino)-1-oxo-3-phenylpropan-2-yl)-6-(2,2,2-t-
rifluoroacetamido)hexanamide hydrochloride (10) (0.33 g, 100%
yield).
##STR00122## To a solution of
(S)-2-amino-N--((S)-1-(methylamino)-1-oxo-3-phenylpropan-2-yl)-6-(2,2,2-t-
rifluoroacetamido)hexanamide hydrochloride (10) (0.33 g, 0.82 mmol)
in THF (20 mL) were added 7 (0.36 g, 0.82 mmol, 1.0 equiv.), HATU
(0.68 g, 1.65 mmol, 2.0 equiv.) and triethylamine (0.54 g, 4.94
mmol, 6.0 equiv.). The reaction was stirred at room temperature for
3 h. A saturated solution of sodium bicarbonate was added. The
product was extracted with ethyl acetate. The combined organic
layers were washed with water, dried avec sodium sulfate, filtered
and concentrated under reduced pressure. The crude material was
purified by silica gel column (prep-HPLC) to afford tert-butyl
((4S,7S,13S)-4-benzyl-13-isobutyl-3,6,9,12,15-pentaoxo-7-(4-(2,2,2-triflu-
oroacetamido) butyl)-2,5,8,11,14-pentaazaicosan-20-yl)carbamate
(11) (0.35 g, 51% yield).
##STR00123## To a solution of tert-butyl
((4S,7S,13S)-4-benzyl-13-isobutyl-3,6,9,12,15-pentaoxo-7-(4-(2,2,2-triflu-
oroacetamido) butyl)-2,5,8,11,14-pentaazaicosan-20-yl)carbamate
(11) (0.38 g, 0.87 mmol) in 1,4-dioxane (10 mL) and was added a 5M
solution of HCl in 1,4-dioxane (20 mL). The reaction was stirred at
room temperature for 3 h. The reaction was then concentrated and
dried under reduced pressure to afford
(S)-2-(2-((S)-2-(6-aminohexanamido)-4-methylpentanamido)acetami-
do)-N--((S)-1-(methylamino)-1-oxo-3-phenylpropan-2-yl)-6-(2,2,2-trifluoroa-
cetamido)hexanamide (12) (0.33 g, 100% yield).
##STR00124## To a solution of
(S)-2-(2-((S)-2-(6-aminohexanamido)-4-methylpentanamido)acetamido)-N--((S-
)-1-(methylamino)-1-oxo-3-phenylpropan-2-yl)-6-(2,2,2-trifluoroacetamido)h-
exanamide (12) (0.22 g, 0.32 mmol, 1.0 equiv.) in THF (10 mL) at
room temperature was added 5-FAM (13) (0.11 g, 0.32 mmol, 1.0
equiv.), BOP (0.44 g, 1.0 mmol, 3.1 equiv.) and triethylamine (0.6
mL). The reaction was stirred at room temperature for 22 h. The
mixture was then filtered through Celite. The filtrate was
concentrated under reduced pressure. The crude residue was purified
by prep-HPLC to give
5-(4S,7S,13S)-4-benzyl-13-isobutyl-3,6,9,12,15-pentaoxo-7-(4-(2,2,2-trifl-
uoroacetamido)butyl)-2,5,8,11,14-pentaazaicosan-20-yl)carbamoyl)-2-(6-hydr-
oxy-3-oxo-3H-xanthen-9-yl)benzoic acid (14) as a yellow solid (13
mg, 3.9% yield). ESI+ MS: m/z 1044 ([M+H].sup.+), 1H NMR (MeOD, 500
Hz) .delta. 0.92 (d, J=6.5 Hz, 3H), 0.97 (d, J=6.5 Hz, 3H),
1.16-1.19 (m, 2H), 1.27-1.31 (m, 2H), 1.45-1.48 (m, 4H), 1.61-1.71
(m, 8H), 2.31-2.33 (m, 2H), 2.70 (s, 3H), 2.81-2.84 (m, 1H),
2.96-2.98 (m, 1H), 3.17-3.19 (m, 3H), 3.44 (t, J=9.0 Hz, 2H), 3.75
(d, J=15.5 Hz, 1H), 3.92 (d, J=15.5 Hz, 1H), 4.12-4.14 (m, 1H),
4.28-4.30 (m, 1H), 4.50-4.52 (m, 1H), 6.52-6.54 (m, 2H), 6.58 (d,
J=8.5 Hz, 2H), 6.68 (d, J=1.5 Hz, 2H), 7.18-7.29 (m, 6H), 8.18 (d,
J=8 Hz, 1H), 8.42 (s, 1H).
Example 2: Synthesis of
5-(((4S,7S,13S)-7-(4-acetamidobutyl)-4-benzyl-13-isobutyl-3,6,9,12,15-pen-
taoxo-2,5,8,11,14-pentaazaicosan-20-yl)carbamoyl)-2-(6-hydroxy-3-oxo-3H-xa-
nthen-9-yl)benzoic Acid, Broad Substrate C
##STR00125##
The above depicted compound was synthesized using a similar
procedure as Example 1. ESI+MS: m/z 990 ([M+H].sup.+).
Example 3:
2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)-5-(((8S,14S)-1,1,1-trifluor-
o-14-isobutyl-8-((4-methyl-2-oxo-2H-chromen-7-yl)carbamoyl)-2,10,13,16-tet-
raoxo-3,9,12,15-tetraazahenicosan-21-yl)carbamoyl)benzoic Acid,
Broad Substrate B
##STR00126##
The above depicted compound was synthesized using a similar
procedure as Example 1. ESI+MS: m/z 1041 ([M+H].sup.+).
Example 4:
2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)-5-(((8S,14S)-14-isobutyl-8--
((4-methyl-2-oxo-2H-chromen-7-yl)carbamoyl)-2,10,13,16-tetraoxo-3,9,12,15--
tetraazahenicosan-21-yl)carbamoyl)benzoic Acid, Broad Substrate
A
##STR00127##
The above depicted compound was synthesized using a similar
procedure as Example 1. ESI+MS: m/z 1010 ([M+Na].sup.+).
Example 5: Determination of HDAC Activity and Inhibitory Activity
of Reference Compounds Using Compounds of Formula I
Materials and Instrument
All HDACs were purchased from BPS Bioscience. HDAC substrate H218
was from Caliper Co. and Peptides Broad Substrate A, Broad
Substrate E, Broad Substrate B were synthesized in house. The other
reagents purchased from Sigma. Caliper EZ reader II system was used
to collect all the data.
To test HDAC enzymatic activity, purified HDACs were incubated with
2 .mu.M carboxyfluorescein (FAM)-labeled acetylated peptide
substrate for various time at room temperature, in HDAC assay
buffer that contained 50 mM HEPES (pH 7.4), 100 mM KCl, 0.01% BSA
and 0.001% Tween-20. Reactions were terminated by the addition of
the known HDAC inhibitor LBH -589 (panobinostat) with a final
concentration of 1.5 .mu.M. Substrate and product were separated
electrophoretically and fluorescence intensity in the substrate and
product peaks was determined and analyzed by Labchip EZ Reader II.
The percentage of substrate conversion was used for HDAC activity
comparison.
To test inhibitory activity of reference compounds to HDACs,
purified HDACs were incubated with 2 .mu.M carboxyfluorescein
(FAM)-labeled acetylated peptide substrate and tested compound at
varying doses for one hour at room temperature, in HDAC assay
buffer. Reactions were terminated by the addition of the known HDAC
inhibitor LBH -589 (panobinostat) with a final concentration of 1.5
.mu.M. The percent inhibition was plotted against the compound
concentration and the IC50 value was determined from the logistic
dose-response curve fitting by Origin 8.0 software. (Madan
Katragadda, Paola Magotti, Ga. Sfyroera, and John D. Lambris, J.
Med. Chem. 2006, 49, 4616-4622). The reactions were performed in
duplicate for each sample.
Example 6: Kinetics of the Inhibition of HDAC 1 with its
Inhibitors
The kinetics of the inhibition of HDAC1 with SAHA or CI-994 was
measured with Broad Substrate A on Caliper EZ reader II system. To
establish the time-dependent mechanism of inhibition, the progress
curves for HDAC1 in the presence of increasing concentrations of
inhibitor SAHA or CI-994 were monitored for 4 hours. To test the
reversibility of SAHA or CI-994, HDAC1 at 100-fold its final assay
concentration (100 nM) and inhibitor at 10.about.20 fold its
IC.sub.50 after 1 h preincubation was diluted 100-fold with assay
buffer containing 2 .mu.M Broad Substrate A. Substrate conversion
was monitored continuously on EZ reader II. Kinetic parameters
(kon, koff) were derived from slow binding equations known in the
art (See e.g., Chou C. J., et al., J. Biol. Chem., 2008, 283,
35402-35409). SAHA was determined to be a fast on/fast off
inhibitor for HDAC1 (FIG. 6) and CI-994 was determined to be a slow
on/slow off inhibitor for HDAC1 (FIG. 7). The table below shows a
summary of the kinetic parameters for SAHA and CI-994:
TABLE-US-00006 Kinetic Parameters Summary CI-994 SAHA HDAC1
Kon(min-1, .mu.M-1) 0.25 Koff(min-1) 0.0094 >0.2 T(1/2) min 74
<4 Ki(nM) 37 ~1.9** IC50(nM) @3 hr 46 5 **Ki was estimated from
HDAC1 stability in the presence of SAHA
Structures of the reference compounds are given below:
##STR00128## The above reference compounds are discussed in Wilson
A. J.; Holson, E.; Wagner, F.; Zhang, Y.-L.; Fass, D. M.; Haggarty,
S. J.; Bhaskara, S.; Hiebert, S. W.; Schreiber, S. L.; Khabele, D.
Cancer Biology & Therapy, 2011, Volume 12 Issue 6, 1-10: "The
DNA damage mark pH2AX differentiates the cytotoxic effects of small
molecule HDAC inhibitors in ovarian cancer cells".
While this invention has been particularly shown and described with
references to preferred embodiments thereof, it will be understood
by those skilled in the art that various changes in form and
details may be made therein without departing from the scope of the
invention encompassed by the appended claims.
SEQUENCE LISTINGS
1
2915PRTArtificial SequenceSynthetic PeptideMOD_RES(1)..(4)absent or
a natural or unnatrural amino acid 1Xaa Xaa Xaa Xaa Lys 1 5
25PRTArtificial SequenceSynthetic PeptideMOD_RES(1)..(4)absent or a
natural or unnatrural amino acidMOD_RES(5)..(5)Ac = Lys 2Xaa Xaa
Xaa Xaa Lys 1 5 35PRTArtificial SequenceSynthetic
PeptideMOD_RES(1)..(4)absent or a natural or unnatrural amino
acidMOD_RES(5)..(5)COCF3 = Lys 3Xaa Xaa Xaa Xaa Lys 1 5
47PRTArtificial SequenceSynthetic PeptideMOD_RES(4)..(4)Ac = Lys
4Thr Gly Gly Lys Ala Pro Arg 1 5 57PRTArtificial SequenceSynthetic
PeptideMOD_RES(3)..(3)Ac- LysMOD_RES(7)..(7)Ac- Lys 5Leu Gly Lys
Gly Gly Ala Lys 1 5 67PRTArtificial SequenceSynthetic
PeptideMOD_RES(6)..(7)Ac = Lys 6Thr Ser Pro Gln Pro Lys Lys 1 5
76PRTArtificial SequenceSynthetic PeptideMOD_RES(5)..(6)Ac = Lys
7Ser Pro Gln Pro Lys Lys 1 5 85PRTArtificial SequenceSynthetic
PeptideMOD_RES(4)..(5)Ac = Lys 8Pro Gln Pro Lys Lys 1 5
95PRTArtificial SequenceSynthetic PeptideMOD_RES(5)..(5)Ac = Lys
9Thr Ser Arg His Lys 1 5 107PRTArtificial SequenceSynthetic
PeptideMOD_RES(4)..(4)COCF3 = Lys 10Thr Gly Gly Lys Ala Pro Arg 1 5
117PRTArtificial SequenceSynthetic PeptideMOD_RES(3)..(3)COCF3 =
LysMOD_RES(7)..(7)COCF3 = Lys 11Leu Gly Lys Gly Gly Ala Lys 1 5
127PRTArtificial SequenceSynthetic PeptideMOD_RES(6)..(7)COCF3 =
Lys 12Thr Ser Pro Gln Pro Lys Lys 1 5 136PRTArtificial
SequenceSynthetic PeptideMOD_RES(5)..(6)COCF3 = Lys 13Ser Pro Gln
Pro Lys Lys 1 5 145PRTArtificial SequenceSynthetic
PeptideMOD_RES(4)..(5)COCF3 = Lys 14Pro Gln Pro Lys Lys 1 5
155PRTArtificial SequenceSynthetic PeptideMOD_RES(5)..(5)COCF3 =
Lys 15Thr Ser Arg His Lys 1 5 164PRTArtificial SequenceSynthetic
PeptideMOD_RES(3)..(4)Ac = Lys 16Arg His Lys Lys 1 174PRTArtificial
SequenceSynthetic PeptideMOD_RES(3)..(4)Ac = Lys 17Gln Pro Lys Lys
1 184PRTArtificial SequenceSynthetic PeptideMOD_RES(3)..(4)COCF3 =
Lys 18Arg His Lys Lys 1 194PRTArtificial SequenceSynthetic
PeptideMOD_RES(3)..(4)COCF3 = LysMOD_RES(3)..(4)COCF3 = Lys 19Gln
Pro Lys Lys 1 204PRTArtificial SequenceSynthetic Peptide 20Arg His
Lys Lys 1 214PRTArtificial SequenceSynthetic Peptide 21Gln Pro Lys
Lys 1 227PRTArtificial SequenceSynthetic Peptide 22Thr Gly Gly Lys
Ala Pro Arg 1 5 237PRTArtificial SequenceSynthetic Peptide 23Leu
Gly Lys Gly Gly Ala Lys 1 5 247PRTArtificial SequenceSynthetic
Peptide 24Thr Ser Pro Gln Pro Lys Lys 1 5 256PRTArtificial
SequenceSynthetic Peptide 25Ser Pro Gln Pro Lys Lys 1 5
265PRTArtificial SequenceSynthetic Peptide 26Pro Gln Pro Lys Lys 1
5 275PRTArtificial SequenceSynthetic Peptide 27Thr Ser Arg His Lys
1 5 289PRTArtificial SequenceSynthetic PeptideMOD_RES(9)..(9)Ac =
Lys 28Phe Ala Met Ala His Ala Leu Gly Lys 1 5 299PRTArtificial
SequenceSynthetic Peptide 29Phe Ala Met Ala His Ala Leu Gly Lys 1
5
* * * * *